Enrichment of hexachlorobenzene and 1,3,5-trichlorobenzene transforming bacteria from sediments in Germany and Vietnam

Bacterial cultures were enriched from sediments in Germany and Vietnam reductively dechlorinating hexachlorobenzene and the highly persistent 1,3,5-trichlorobenzene to monochlorobenzene. The main products of the reductive dechlorination of hexachlorobenzene were monochlorobenzene and dichlorobenzenes (1,2-; 1,3- and 1,4-dichlorobenzene) while no trichlorobenzenes accumulated. For the reductive dechlorination of 1,3,5-trichlorobenzene with the mixed culture from Vietnam sediment, 1,3- dichlorobenzene and monochlorobenzene were produced as intermediate and final end-product, respectively. The pattern of dechlorination did not change when the cultures were repeatedly exposed to oxygen over seven transfers demonstrating oxygen tolerance of the dechlorinating bacteria. However, reductive dechlorination of 1,3,5-trichlorobenzene was inhibited by vancomycin at a concentration of 5 mg L^?1. Vancomycin delayed reductive dechlorination of hexachlorobenzene in mixed cultures by about 6 months. When repeatedly applied, vancomycin completely abolished the ability of the mixed culture to transform hexachlorobenzene. Sensitivity to vancomycin and insensitivity to brief exposure of oxygen indicates that the dechlorinating bacteria in the mixed cultures did not belong to the genus Dehalococcoides.     

Heavy metal tolerance in Festuca ovina L. from contaminated sites in the Eifel Mountains,Germany

Extremely high degrees of lead tolerance, measured by comparing rates of root extension in culture solutions, are reported from populations of Festuca ovina growing at two lead-mining sites (Westschacht and Keldenich-II) near Mechernich in the Eifel Mountains, Germany. Other populations from nearby heavy metal-contaminated areas show a considerably smaller degree of lead tolerance. Samples of Festuca ovina collected in the field at Westschacht and Keldenich-II contain higher levels of lead in their aerial organs than do those from other lead-contaminated sites. The main soil factor determining the high degree of lead tolerance is the high Pb/Ca ratio. Populations from soils with a low Pb/Ca ratio display a very low degree of tolerance. It is therefore concluded that in Westschacht and Keldenich-II plants, a genuine intracellular tolerance mechanism is present, allowing the accumulation of lead in aerial organs.Leaf samples of zinc-tolerant Festuca contain higher levels of zinc than do samples of non-tolerant plants. Lead and zinc amounts in leaves are correlated with the soil ratios of Pb/Ca and Zn/Ca, respectively, rather than with the absolute soil-metal levels.In a slightly lead-tolerant, but highly zinc-tolerant clone of Festuca ovina from a site contaminated with large amounts of lead and zinc (Plombières), lead was found to be the major factor affecting the inhibition of root extension with combined treatments of lead and zinc in culture solutions. In the highly lead-tolerant, zinc-sensitive population from Westschacht, zinc governs the response of root growth to combinations of the two metals. The results are discussed in terms of discriminating distinct types of heavy-metal tolerance.     

Development of a novel technique for axenic isolation and culture of thraustochytrids from New Zealand marine environments

Aims: To maintain axenic cultures of commercially important thraustochytrids, a novel procedure was developed for the isolation of zoospores and sporangium from heterotrophic seawater samples and axenic culture on solid media. Methods and Results: Thraustochytrid cultures were isolated from Whangapoua Harbour in North East New Zealand and subjected to two antibiotic and antifungal treatment regimes designed to eliminate bacteria and fungi. Antibiotic trial 1 was designed to determine the appropriate combination of antibiotics (including streptomycin/penicillin, ampicillin, rifampicin, nalidixic acid, tetracycline, gentamicin and the antifungal agent nystatin). Antibiotic trial 2 determined the optimal dosing frequency and concentration of the antibiotics, and antifungal found to be the most promising in trial 1. Axenic cultures were then spread plated onto nutrient agar containing the optimal antibiotic cocktail, and pure thraustochytrid colonies were purified on solid media using standard microbiological techniques. Conclusions: Removal of bacteria and fungi was best accomplished using a mixture of three antibiotics and one antifungal; rifampicin (300 mg l^?1), streptomycin/penicillin (25 mg l^?1) and nystatin (10 mg l^?1) were incorporated in seawater samples and incorporated into cultures every 24 h for a minimum of 2 days. Significance and Impact of the Study: The axenic isolation and culture of marine thraustochytrids from a marine habitat in New Zealand have significant implications for the biotechnological development of these potentially valuable protists. This method has global significance as it is reasonable to assume it could be used throughout the world to obtain axenic thraustochytrid cultures.     

Enumeration and characterization of nitrogen-fixing bacteria in an eelgrass (Zostera marina) bed

Marine nitrogen-fixing bacteria distributed in the eelgrass bed and seawater of Aburatsubo Inlet, Kanagawa, Japan were investigated using anaerobic and microaerobic enrichment culture methods. The present enrichment culture methods are simple and efficient for enumeration and isolation of nitrogen-fixing bacteria from marine environments. Mostprobable-number (MPN) values obtained for nitrogen-fixing bacteria ranged from 1.1×10² to 4.6×10²/ml for seawater, 4.0×10⁴ to 4.3×10⁵/g wet wt for eelgrass-bed sediment, and 2.1 × 10⁵ to 1.2 × 10⁷/g wet wt for eelgrass-root samples. More than 100 strains of halophilic, nitrogen-fixing bacteria belonging to the family Vibrionaceae were isolated from the MPN tubes. These isolates were roughly classified into seven groups on the basis of their physiological and biochemical characteristics. The majority of the isolates were assigned to the genusVibrio and one group to the genusPhotobacterium. However, there was also a group that could not be identified to the generic level. All isolates expressed nitrogen fixation activities under anaerobic conditions, and no organic growth factors were required for their activities.     

Dynamics of bacterial populations during bench‐scale bioremediation of oily seawater and desert soil bioaugmented with coastal microbial mats

This study describes a bench‐scale attempt to bioremediate Kuwaiti, oily water and soil samples through bioaugmentation with coastal microbial mats rich in hydrocarbonoclastic bacterioflora. Seawater and desert soil samples were artificially polluted with 1% weathered oil, and bioaugmented with microbial mat suspensions. Oil removal and microbial community dynamics were monitored. In batch cultures, oil removal was more effective in soil than in seawater. Hydrocarbonoclastic bacteria associated with mat samples colonized soil more readily than seawater. The predominant oil degrading bacterium in seawater batches was the autochthonous seawater species Marinobacter hydrocarbonoclasticus. The main oil degraders in the inoculated soil samples, on the other hand, were a mixture of the autochthonous mat and desert soil bacteria; Xanthobacter tagetidis, Pseudomonas geniculata, Olivibacter ginsengisoli and others. More bacterial diversity prevailed in seawater during continuous than batch bioremediation. Out of seven hydrocarbonoclastic bacterial species isolated from those cultures, only one, Mycobacterium chlorophenolicum, was of mat origin. This result too confirms that most of the autochthonous mat bacteria failed to colonize seawater. Also culture‐independent analysis of seawater from continuous cultures revealed high‐bacterial diversity. Many of the bacteria belonged to the Alphaproteobacteria, Flavobacteria and Gammaproteobacteria, and were hydrocarbonoclastic. Optimal biostimulation practices for continuous culture bioremediation of seawater via mat bioaugmentation were adding the highest possible oil concentration as one lot in the beginning of bioremediation, addition of vitamins, and slowing down the seawater flow rate.     

Accelerated decolorization of reactive azo dyes under saline conditions by bacteria isolated from Arabian seawater sediment

Presence of huge amount of salts in the wastewater of textile dyeing industry is one of the major limiting factors in the development of an effective biotreatment system for the removal of azo dyes from textile effluents. Bacterial spp. capable of thriving under high salt conditions could be employed for the treatment of saline dye-contaminated textile wastewaters. The present study was aimed at isolating the most efficient bacterial strains capable of decolorizing azo dyes under high saline conditions. Fifty-eight bacterial strains were isolated from seawater, seawater sediment, and saline soil, using mineral salt medium enriched with 100?mg?l^?1 Reactive Black-5 azo dye and 50?g NaCl l^?1 salt concentration. Bacterial strains KS23 (Psychrobacter alimentarius) and KS26 (Staphylococcus equorum) isolated from seawater sediment were able to decolorize three reactive dyes including Reactive Black 5, Reactive Golden Ovifix, and Reactive Blue BRS very efficiently in liquid medium over a wide range of salt concentration (0–100?g NaCl l^?1). Time required for complete decolorization of 100?mg dye l^?1 varied with the type of dye and salt concentration. In general, there was an inverse linear relationship between the velocity of the decolorization reaction (V) and salt concentration. This study suggested that bacteria isolated from saline conditions such as seawater sediment could be used in designing a bioreactor for the treatment of textile effluent containing high concentration of salts.     

Degradation of Putrescine and Cadaverine in Seawater Cultures by Marine Bacteria

Marine bacteria removed two diamines, putrescine and cadaverine, from coastal seawater supplemented only with these compounds. Batch cultures of natural bacterial communities were grown in filtered seawater (0.05 μm) supplemented with 500 μg of putrescine or cadaverine per liter. Increases in bacterial cell number were counted with an epifluorescence microscope after acridine orange staining. Removal of diamines from seawater was monitored by high-performance liquid chromatography. Diamines were removed from the seawater cultures within 48 h with no corresponding increase in bacterial yield, growth rate, or viability relative to control (unsupplemented) cultures. Shipboard experiments with open-ocean deep water (1,500 m) showed similar, if slower, removal of putrescine from seawater. Unlike uptake experiments with amino acids, labeled putrescine experiments indicated that most putrescine carbon is mineralized to CO₂ rather than assimilated by the bacteria. After growth in unsupplemented control cultures, the bacteria showed a significant potential to mineralize putrescine, indicating a general degradation potential for this compound by marine bacteria even if the compound was not present during growth. Indicators of metabolic activity such as glucose and glutamic acid uptake and mineralization were not affected by the presence of putrescine. This shows that at the concentrations added, the diamines are not toxic, and therefore detoxification was not the reason for degradation of the diamines by the bacteria.     

Isolation of Typical Marine Bacteria by Dilution Culture: Growth, Maintenance, and Characteristics of Isolates under Laboratory Conditions

Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coast were cultured in filtered autoclaved seawater following dilution to extinction. The populations present before dilution varied from 0.11 × 10⁹ to 1.07 × 10⁹ cells per liter. The mean cell volume varied between 0.042 and 0.074 μm³, and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DNA per cell. All three parameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from very high dilutions of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria. However, 15 of these isolates were eventually obtained from dilution cultures that could initially be cultured only on very low-nutrient media and that could initially not form visible colonies on any of the agar media tested, indicating that these cultures contained obligately oligotrophic bacteria. It was concluded that the cells in these 15 dilution cultures had adapted to growth under laboratory conditions after several months of nutrient deprivation prior to isolation. From the North Sea experiment, it was concluded that the contribution of facultative oligotrophs and eutrophs to the total population was less than 1% and that while more than half of the population behaved as obligately oligotrophic bacteria upon first cultivation in the dilution culture media, around 50% could not be cultured at all. During one of the Resurrection Bay experiments, 53% of the dilution cultures obtained from samples diluted more than 2.5 × 10⁵ times consisted of such obligate oligotrophs. These cultures invariably harbored a small rod-shaped bacterium with a mean cell volume of 0.05 to 0.06 μm³ and an apparent DNA content of 1 to 1.5 fg per cell. This cell type had the dimensions of ultramicrobacteria. Isolates of these ultramicrobacterial cultures that were eventually obtained on relatively high-nutrient agar plates were, with respect to cell volume and apparent DNA content, identical to the cells in the initially obligately oligotrophic bacterial dilution culture. Determination of kinetic parameters from one of these small rod-shaped strains revealed a high specific affinity for the uptake of mixed amino acids (a°_A, 1,860 liters/g of cells per h), but not for glucose or alanine as the sole source of carbon and energy (a°_A, ± 200 liters/g of cells per h). The ultramicrobial strains obtained are potentially a very important part of picoplankton biomass in the areas investigated.     

Enumeration and Detection of Anaerobic Ferrous Iron-Oxidizing, Nitrate-Reducing Bacteria from Diverse European Sediments

Anaerobic, nitrate-dependent microbial oxidation of ferrous iron was recently recognized as a new type of metabolism. In order to study the occurrence of three novel groups of ferrous iron-oxidizing, nitrate-reducing bacteria (represented by strains BrG1, BrG2, and BrG3), 16S rRNA-targeted oligonucleotide probes were developed. In pure-culture experiments, these probes were shown to be suitable for fluorescent in situ hybridization, as well as for hybridization analysis of denaturing gradient gel electrophoresis (DGGE) patterns. However, neither enumeration by in situ hybridization nor detection by the DGGE-hybridization approach was feasible with sediment samples. Therefore, the DGGE-hybridization approach was combined with microbiological methods. Freshwater sediment samples from different European locations were used for enrichment cultures and most-probable-number (MPN) determinations. Bacteria with the ability to oxidize ferrous iron under nitrate-reducing conditions were detected in all of the sediment samples investigated. At least one of the previously described types of bacteria was detected in each enrichment culture. MPN studies showed that sediments contained from 1 × 10⁵ to 5 × 10⁸ ferrous iron-oxidizing, nitrate-reducing bacteria per g (dry weight) of sediment, which accounted for at most 0.8% of the nitrate-reducing bacteria growing with acetate. Type BrG1, BrG2, and BrG3 bacteria accounted for an even smaller fraction (0.2% or less) of the ferrous iron-oxidizing, nitrate-reducing community. The DGGE patterns of MPN cultures suggested that more organisms than those isolated thus far are able to oxidize ferrous iron with nitrate. A comparison showed that among the anoxygenic phototrophic bacteria, organisms that have the ability to oxidize ferrous iron also account for only a minor fraction of the population.     

BACTERIA THAT INDUCE MORPHOGENESIS IN ULVA PERTUSA (CHLOROPHYTA) GROWN UNDER AXENIC CONDITIONS1

Marine foliaceous green macroalgae such as Ulva lose their typical morphology when cultured aseptically in defined synthetic media. However, after reinfection by certain marine bacteria (isolated from unialgal cultures of Ulva pertusa Kjellman), the organisms regain their typical foliaceous or tubular morphology. To investigate the morphogenesis (MG) induced in U. pertusa by bacteria, we isolated and identified bacteria with MG activity on U. pertusa and studied the distribution of such bacteria in seawater and on various marine macroalgae. We isolated 1555 bacterial strains from 18 species of marine macroalgae (six Chlorophyta, five Phaeophyta, and seven Rhodophyta), from seawater and from sediment collected at the beach at Omaezaki, Shizuoka Prefecture; Japan. Of these, 676 bacterial strains (43.5%) showed MG activity. They were classified into six bacterial groups, Flavobacterium, Vibrio, Pseudomonas, Deleya, Escherichia, and gram-positive cocci. These bacteria were ubiquitous among the samples and were not specific to U. pertusa. Several plant growth regulators had no MG activity. Filter-sterilized supernatants of culture media of MG-active bacteria strains did not induce MG. Cocultivation of Ulva with active bacterial strains is so far the only way to induce the MG effect, which suggests that for MG direct contact between Ulva and the bacterial strain is necessary.     

A new insight into lead (II) tolerance of environmental fungi based on a study of Aspergillus niger and Penicillium oxalicum

Environmental microorganisms have been widely applied in heavy metal remediation. This study explored the mechanisms of lead tolerance of two typical filamentous fungi, Aspergillus niger and Penicillium oxalicum. It is shown that the mechanisms of reducing Pb toxicity by these two fungi have three major pathways. The secreted oxalic acid can react with Pb (II) to form insoluble Pb minerals, primarily lead oxalate. Then, the enhanced biosorption via forming new border of cell wall prevents the transportation of Pb (II) into hypha. In addition, the fungal activity could be maintained even at high Pb concentration due to the intracellular accumulation. It was confirmed that A. niger has the higher Pb tolerance (up to 1500 mg l⁻¹ Pb level) compared with P. oxalicum (up to 1000 mg l⁻¹). Meanwhile, Pb levels below 1000 mg l⁻¹ partially stimulate the bioactivity of A. niger, which was confirmed by its elevated respiration (from 53 to 63 mg C l⁻¹ medium h⁻¹). This subsequently enhanced microbial functions of A. niger to resist Pb toxicity. A better understanding of Pb tolerance of these two fungi sheds a bright future of applying them to remediate lead-contaminated environments.     

Amendment Optimization to Enhance Lead Extractability from Contaminated Soils for Phytoremediation

Eight lead-contaminated soils and one background soil artificially contaminated with several lead compounds were examined to determine the factors that limit lead extractability and thus plant availability during phytoremediation, as lead must be in soluble form for plant uptake to occur. The effect of the chemical form of the lead as well as the association of the lead among the different soil chemical fractions on lead extractability was specifically addressed. Results indicate that all the added lead forms tested except PbCrO₄ were readily extracted and believed to be available for plant uptake, operationally defined as EDTA-extractable lead, as EDTA is the primary soil amendment for phytoremediation of lead-contaminated soils. Sequential extraction of the eight lead-contaminated soils that previously had been extracted with EDTA shows that the EDTA-extractable or plant available lead corresponds to mainly the exchangeable and carbonate fractions of each soil. Lead associated with oxide, organic, and residual fractions were less effectively targeted and solubilized by EDTA and therefore are not as readily available for plant uptake. Attempts to increase the available pool of soluble lead included the combination of EDTA with organic acids, reducing agents, and surfactants. Results from these studies indicate that high concentrations or extremely low pH conditions are required to enhance the plant available pool of lead by the organic acids and reducing agents. Surfactants, particularly caprylic acid in combination with 0.25 mM EDTA, were shown to be as effective as 0.50 mM EDTA alone. An amendment formulation combining less EDTA with surfactants is attractive for phytoremediation because of the biodegradability and cost concerns commonly associated with using larger amounts of EDTA.     

Mechanisms and Rates of Decay of Marine Viruses in Seawater

Loss rates and loss processes for viruses in coastal seawater from the Gulf of Mexico were estimated with three different marine bacteriophages. Decay rates in the absence of sunlight ranged from 0.009 to 0.028 h^-1, with different viruses decaying at different rates. In part, decay was attributed to adsorption by heat-labile particles, since viruses did not decay or decayed very slowly in seawater filtered through a 0.2-μm-pore-size filter (0.2-μm-filtered seawater) and in autoclaved or ultracentrifuged seawater but continued to decay in cyanide-treated seawater. Cyanide did cause decay rates to decrease, however, indicating that biological processes were also involved. The observations that decay rates were often greatly reduced in 0.8- or 1.0-μm-filtered seawater, whereas bacterial numbers were not, suggested that most bacteria were not responsible for the decay. Decay rates were also reduced in 3-μm-filtered or cycloheximide-treated seawater but not in 8-μm-filtered seawater, implying that flagellates consumed viruses. Viruses added to flagellate cultures decayed at 0.15 h^-1, corresponding to 3.3 viruses ingested flagellate^-1 h^-1. Infectivity was very sensitive to solar radiation and, in full sunlight, decay rates were 0.4 to 0.8 h^-1. Even when UV-B radiation was blocked, rates were as high as 0.17 h^-1. Calculations suggest that in clear oceanic waters exposed to full sunlight, most of the virus decay, averaged over a depth of 200 m, would be attributable to solar radiation. When decay rates were averaged over 24 h for a 10-m coastal water column, loss rates of infectivity attributable to sunlight were similar to those resulting from all other processes combined. Consequently, there should be a strong diel signal in the concentration of infectious viruses. In addition, since sunlight destroys infectivity more quickly than virus particles, a large proportion of the viruses in seawater is probably not infective.     

Growth responses of ciliate protozoa to the abundance of their bacterial prey

The growth rate or numerical response of five species of bactivorous ciliates to the abundance ofEnterobacter aerogenes was examined in monoxenic culture. The ciliatesColpidium campylum, C. colpoda, Glaucoma scintillons, G. frontata, andCyclidium glaucoma were isolated from a small pond. Four were grown in shaken cultures, while three were grown in cultures in which the bacteria were allowed to settle on the bottom of the culture vessel. Of the seven response curves generated, four had distinct thresholds, so that the Michaelis-Menten model usually fitted to ciliate numerical response curves was not appropriate. In shaken cultures, half-saturation prey densities ranged from 5.5 × 10⁶ to 42.9 × 10⁶ bacteria/ml. In unshaken cultures, half-saturation densities ranged from 0.057 × 10⁶ to 14.6 × 10⁶ bacteria/cm². Two species grown on both suspended and settled bacteria attained higher growth rates and had lower half-saturation prey densities feeding on settled bacteria.     

Response of bacteria and phytoplankton to contaminated sediments from Trenton Channel,Detroit River

Several types of bioassays were used in 1986 and 1987 to investigate the effect of contaminated sediments on natural populations of bacteria and phytoplankton from the Trenton Channel, Detroit River. The approach included the measurement of uptake of ³H-glucose or ³H-adenine by bacteria and ¹⁴C-bicarbonate by phytoplankton in the presence of different amounts of Trenton Channel and Lake Michigan (control) sediments. Trenton Channel sediments are contaminated by high levels of toxic organic compounds and metals, especially zinc, lead, and copper. Because levels of biomass of bacteria and phytoplankton varied widely among the different bioassays, it was necessary to adjust uptake rates for biomass. Biomass adjustments were made using acridine orange counts for bacteria and chlorophyll measurements for phytoplankton. The results show a statistically significant suppression of uptake of substrates for both bacteria and phytoplankton with increasing amounts of sediment. Uptake was suppressed as much as 90 percent for bacteria and 93 percent for phytoplankton at 1200 mg l^-1 of Trenton Channel sediments compared to bioassays without sediment. Uncontaminated Lake Michigan sediment suppressed uptake much less than Detroit River sediment; the difference in suppression of uptake between the two types of sediment was statistically significant for both bacteria and phytoplankton.Contribution No. 518 of the Center for Great Lakes and Aquatic Sciences of the University of Michigan.     

Bacterial Growth on Coral Mucus

Coral mucus-degrading bacteria were isolated by an enrichment culture procedure. The isolates were able to grow as pure cultures on 10% sterilized mucus in seawater, yielding 10⁸ CFU/ml. The isolates, mostly Vibrio strains, were classified by classical and molecular methods. When carbon, nitrogen, and phosphorus compounds were added separately to the mucus medium, there was no increase in CFUs; however, when they were added together, there was a large increase in cell yield. The indigenous bacterial population of coral mucus increased from 10³ to 10⁸ CFU/ml when incubated at 30°C for 11 h, changing from a heterogeneous community to a Vibrio-dominated population. Factors which regulate the abundance and diversity of coral mucus bacteria are discussed.     

Vertical distribution in and isolation of bacteria from Lake Vanda: an Antarctic lake

Vertical distribution of bacteria in Lake Vanda, an Antarctic meromictic lake, was examined by the acridine orange epifluorescence direct count method. Total bacteria were 10⁴–10⁵ cells · ml^–1 in the water at 55 m depth and above, and increased drastically to 10⁷ cells · ml^–1 in the bottom water. Filamentous or long rodshaped bacteria occurred at a high frequency in the upper layers, but in the bottom layers most bacteria were coccoidal or short rods. Mean bacterial cell volume in water of between 10 m and 60 m deep was fairly large compared with common bacterial populations in seawater and lake water. Aerobic heterotrophic bacteria were recovered from the water of a depth of 30 m and above, and were assumed to belong to Caulobacter. Viable heterotrophic bacteria were not recovered from the high salinity deep water by media prepared with the same deep water. Phototrophic purple non-sulphur bacteria were isolated by enrichment cultures from water at 55 m depth.     

High Risk of Lead Contamination for Scavengers in an Area with High Moose Hunting Success

Top predators and scavengers are vulnerable to pollutants, particularly those accumulated along the food chain. Lead accumulation can induce severe disorders and alter survival both in mammals (including humans) and in birds. A potential source of lead poisoning in wild animals, and especially in scavengers, results from the consumption of ammunition residues in the tissues of big game killed by hunters. For two consecutive years we quantified the level lead exposure in individuals of a sentinel scavenger species, the common raven (Corvus corax), captured during the moose (Alces alces) hunting season in eastern Quebec, Canada. The source of the lead contamination was also determined using stable isotope analyses. Finally, we identified the different scavenger species that could potentially be exposed to lead by installing automatic cameras targeting moose gut piles. Blood lead concentration in ravens increased over time, indicating lead accumulation over the moose-hunting season. Using a contamination threshold of 100 µg.L⁻¹, more than 50% of individuals were lead-contaminated during the moose hunting period. Lead concentration was twice as high in one year compared to the other, matching the number of rifle-shot moose in the area. Non-contaminated birds exhibited no ammunition isotope signatures. The isotope signature of the lead detected in contaminated ravens tended towards the signature from lead ammunition. We also found that black bears (Ursus americanus), golden eagles and bald eagles (Aquila chrysaetos and Haliaeetus leucocephalus, two species of conservation concern) scavenged heavily on moose viscera left by hunters. Our unequivocal results agree with other studies and further motivate the use of non-toxic ammunition for big game hunting.     

A METHOD FOR OBTAINING AXENIC ALGAL CULTURES USING THE ANTIBIOTIC CEFOTAXIME WITH EMPHASIS ON CLADOPHOROPSIS MEMBRANACEA (CHLOROPHYTA)1

Axenic cultures of the tropical green seaweed Cladophoropsis membranacea (C. Agardh) Boergesen were obtained by cutting 2-mm apical tips from fast-growing unialgal filaments and incubating them in 1/2PES containing 100 μg · mL^?1 cefotaxime. After 1 week, apical tips were cut from the newly grown plantlets, washed through a series of sterile drops of seawater, and incubated in sterile 1/2PES. Plantlets were screened for the presence of bacteria by incubating droplets of the culture medium on peptone agar plates and by examining DAPI-stained filaments. Ninety to one hundred percent of the plantlets obtained with this treatment were axenic. Cefotaxime was also effective against cyanobacteria but of only limited value in rhodophytes.     

Enhanced secondary metabolite (tanshinone) production of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> hairy roots in a novel root–bacteria coculture process

Salvia miltiorrhiza Bunge (Lamiaceae) hairy root cultures were inoculated (at 0.02 and 0.2% v/v) and co-cultured with Bacillus cereus bacteria. The root biomass growth was inhibited significantly by the bacteria inoculated to the root culture on the first day (day 0) but not by the bacteria inoculated on days 14 or 21 (in a 28-day overall period). On the other hand, the growth of the bacteria in the hairy root culture was also strongly inhibited by the hairy roots, partially because of the antibacterial activity of the secondary compounds produced by the roots. Most interestingly, the tanshinone production was promoted by the inoculation of bacteria at any of these days but more significantly by an earlier bacteria inoculation. With 0.2% bacteria inoculated on day 0, for example, the total tanshinone content of roots was increased by more than 12-fold (from 0.20 to 2.67 mg g⁻¹ dry weight), and the volumetric tanshinone yield increased by more than sixfold (from 1.40 to 10.4 mg l⁻¹). The tanshinone production was also stimulated by bacterial water extract and bacterial culture supernatant but less significantly than by the inoculation of live bacteria. The results suggest that the stimulation of tanshinone production by live bacteria in the root cultures may be attributed to the elicitor compounds originating from the bacteria, and the hairy root–bacteria coculture may be an effective strategy for improving secondary metabolite production in plant tissue cultures.